Nonradioactive labels

Isotopic tracers are not exclusively radioactive. For instance, is a nonradioactive element that is suitable for a nonradioactive labeling technique. 

Bayer, E.A., and Wilchek, M. (1992) Labeling and detection of proteins and glycoproteins. In Nonradioactive Labeling and Detection of Biomolecules (C. Kessler, ed.), pp. 98-99. Springer-Verlag, New York. 

Kumar, A., Tchen, P., Roullet, F., and Cohen, J. (1988) Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase. Anal. Biochem. 169, 376-382. 

In situ hybridization techniques are used to subtype the papilloma virus that may be found in premalignant lesions in uterine cervix. Most of the techniques use nonradioactively labeled avidin-biotin probes. A number of specific biotin-labeled probe cocktails are available for HPV subtype identification (e.g., HPV 6-l l, 16-18, 31-33-35). Some of these techniques use chemiluminescent components to enhance the sensitivity of HPV subtype detection (H5). 

Schmitz, G. G. Walter, T. Seibl, R. Kessler, C. Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 1991,192(1), 222-231. 

Kessler, C., ed. (1992) Nonradioactive Labeling and Detection of Biomolecules, Springer-Verlag, New York... 

Egger, D, Troxler, M., and Bienz, K (1995) Light- and electron microscopic in situ hybridization- nonradioactive labeling and detection, double hybridization, and combined hybridization—immunocytochemistry J Histochem Cytochem 42, 815-822... 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. and Leary et al. in 1981, the procedure is probably the most popular nonradioactive labeling technique reported for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported in the literature, by far the most frequently employed derivative is biotin—dUTP prepared from the reaction of an amine-modified dUTP with an amine-reactive biotinylation reagent, such as NHS-LC—biotin (Chapter 8, Section 3.1). 

Kessler, C., Holtke, H.-J., Seibl, R., Burg, J., and Muhlegger, K. (1990) Nonradioactive labeling and detection of nucleic acids I. A novel DNA labeling and detection system based on digoxigenimantidigoxigenin ELISA principle (digoxigenin system). Mol. Gen. Hoppe-Seyler 371, 917-927. 

Kumar A, Tchen P, Roullet F, Cohen J. Nonradioactive labeling of synthetic ohgonucletide probes with terminal deoxynucleotidyl transferase. Analyt. Biochem. 1988 169 376-382. 

PEA is an important discovery in the characterization of phosphopantetheinylated proteins. It allows one to analyze nonradioactively labeled substrates thereby making many more substrates accessible to biosynthetic studies of these types of proteins. In addition, this approach often eliminates the need for preparing synthetic standards for comparison of hydrolyzed thioester intermediates, which may be challenging and time consuming to accomplish. Moreover, PEA immediately shows that the substrate or intermediate is connected to the PPant posttranslational modification and not elsewhere on the protein. This is not possible to detect with radioactivity or any other... 

The nature and detection of the different labels, followed by the methods of their incorporation in probes, is reviewed here. Radioactive and nonradioactive labels are not distinguished with respect to the labeling method but differences in methodology are indicated when necessary. 

Radioactive or nonradioactive labels safety, stability, detectability, sensitivity, cost, effect on hybridization kinetics, intrinsic specific activity of precursor... 

Secondary, nonradioactive labels are usually bulky nucleotide analogues which are less good substrates for most polymerases. For instance, BIO-4-dUTP (biotin attached with a 4-atom spacer arm to C-5 of dUTP Fig. 7.10) is a much better substrate than BIO-16-dUTP. On the other hand, the biotin binding sites are deeply buried in two pairs in the tetrameric avidin molecule. This explains why BIO-4-dUMP residues in DNA, in contrast to those with longer spacer arms, bind poorly to avidin or streptavidin (Leary et al., 1983) whereas antibodies to biotin bind to all BlO-nucleotide analogues equally well. Optimum binding is achieved when about 30 base analogues are introduced per kilobase, otherwise either decreased specific activity or decreased duplex stability is observed. 

Incorporation of secondary labels by random primer extension The protocol for the incorporation of secondary, nonradioactive labels differs in several respects from that for radiolabels (Table 7.16B) since (i) tagged precursor dNTPs can be bulky and less suitable as a substrate and (ii) the label density usually should be less (Section 7.6.1.2). In contrast to the protocol of radiolabeling, the concentration of each dNTP largely exceeds its although one of the dNTPs is partially replaced by labeled precursor. Consequently, it is easier to make large quantities without decreasing the specific activity. 

Repair with Klenow fragment by adding 10 pi of Klenow mixture (as prepared in Table 7.16A, step 1) to 10 pi of digested DNA. Proceed with the reaction and extraction as described in Table 7.16A, steps 3 and 4. Nonradioactive labels can be incorporated as described in Table 7.16B. 

A variety of nonradioactive labels can be introduced into primers (Section 6.4) and then used in conventional PCR to incorporate these labels into probes. These labels should be introduced to the 5 end of the primer since for most labels, their attachment to the 3 termini would preclude PCR amplification. 

Hybridization conditions vary with the nature of the target (RNA, DNA), of the probe (polynucleotide versus oligomer radiolabel versus nonradioactive labels RNA versus DNA ds versus ss) and of the membrane. The impact of the different parameters will be reviewed to facilitate the optimization of hybridization. 

A metal-dye detection system has been developed that permits picomolar-range HPLC analysis of inositol phosphates from nonradioactively labeled tissues (Mayr, 1990), and was applied for the determination of masses of inositol phosphates in resting and stimulated skeletal muscles (Mayr and Thieleczek, 1991). 

For nonoligonucleotide probes, in vitro transcription (riboprobes), random-primed labeling, nick translation, and PCR-amplification (all for DNA probes) could be the methods of choice for probe labeling with either radioactive or non-radioactive labels. Unless target sequences are not abundant (neuropeptide mRNAs usually are abundant), the use of nonradioactively labeled probes is highly recommended because of a Speed and ease of detection b. Supenor resolution ... 

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